The possible role of GM technology in the breeding, propagation and processing of future

biomass crops

LÁSZLÓ MÁRTON, MIKLÓS GÁBOR FÁRI Introduction

Interest towards biomass crops (after the beginning of the oil era) dates back to the 1970s, when the first oil crisis hit the oil and carbon energy-dependent modern world.

The recent interest is mainly due to environmental considerations in addition to the increase of oil prices and lessening resources. The role of CO₂ emission accompanying the use of fossil fuels in increasing atmospheric CO₂ concentration is obvious, and its environmental consequences in the climate are also clearly detectable (diminishing ice caps and glaciers, increase in the temperature of oceans, dying coral reefs, increasing sea level etc.).

In geological terms, there is no time for introducing positive changes aimed at stopping or reversing the negative trend. One generation from now, 40–50% more people should be fed and provided with energy, whereas (if we do not make every effort) the average surface temperature could increase by 2–6 Co_{, which} could have dramatic effects such as desertification and a significant reduction and relocation/realignment of the currently used agricultural lands. By maximum use of green energy sources (wind, water, flux and reflux of rivers, solar cells or even nuclear energy) CO₂ emission could be significantly reduced; however, that would not be enough to stop the current trends, and increased fixation and fossilization of CO₂ are also necessary. The most straightforward method for this is by photosynthesis of plants. Field crop production is based mainly on annual plants, which does not reduce the CO₂ level, as the intensive soil cultivation mobilizes more CO₂ than is fixed. Unfortunately, in order to avoid food shortage the current range of agricultural crops cannot be changed much. However, if this greenest energy transformation, namely photosynthesis is used, and it is done with perennial plants where the above-surface parts can serve as energy sources, while the below-surface parts significantly contribute to organic matter (carbon) accumulation in the soil, thereby providing a negative carbon balance, significant atmospheric carbon depletion can be achieved. Plant photosynthesis is the most efficient process for CO₂ removal, which is fuelled by 150 000 TW solar energy hitting the surface of the Earth every year. Presently, we use 15 TW energy yearly, which may reach 20 TW by 2030.

The series of energy conversions in photosynthesis are intensively studied not only for CO₂ fixation and biomass production, which is a central area, but also for more direct utilizations such as direct electricity and H₂ production (natural fuel cells) in artificial and semi-artificial systems as well as for direct production of liquid fuels.

János Gundel

10. The possible role of GM technology in the breeding, propagation and processing of future biomass crops

In the present transitional period, all opportunities must be seized to start positive trends and to avoid an environmental catastrophe. The use of GM technologies cannot be given up for some sentimental principles, since via its use the sustainable approach of the “green” era can be made more diverse and more effective, and giving it up would mean that we give up the most effective, scientifically-proven and rapidly applicable tool for food production and energy production based on green biomass.

The ratio of available marginal lands and the summary of current technologies

In 2010, environmental and agricultural experts determined that there are about 320–702 million hectares of so-called marginal areas in Europe, the USA, Africa, India and South-America, where agricultural production was abandoned or whose fertility is so low that they are unsuitable for profitable agricultural production in their present state. If the steppes, savannas and natural bushy areas (so-called LIHD areas) are added to the marginal lands, then they total 1.107–1.411 million hectares. In theory, agricultural fuel production based on biomass in these lands (using traditional crops and agro-techniques) could produce 26–55% of the world’s liquid fuel demand. Responsible professionals and non-professionals agree that the natural resources accumulated during millions of years cannot be further destroyed, not even in the present intensive era of energy demand.

As a result of the above theoretical calculations, agro-economists drew the attention of the scientific world to the fact that the further demolition of natural vegetation can only be avoided if the world’s energy requirement is produced on only the 320–702 million hectares of marginal agro-ecosystems. For this purpose, as stated by American scientists, the most effective biological, genetic and other breeding methods should be used and new and adequate agricultural cultivation and biomass processing methods should be developed. The prediction is that breeding, propagation and growing of the future biomass and energy crops in the next fifty years will be done by applying a combination of the most modern techniques, among which the GM method should also be used.

What should be the future biomass and/or energy crops like?

There is a severe technical problem in biomass crop production. Plant breeding has a history of 10.000 years, but so far the most frequent target has been the fruit or other specific plant parts, and the aim has been to obtain the highest amount of the utilized part from the lowest possible amount of biomass. With respect to energy, these parts represent a negligible amount, as plants store the chemical energy from CO₂ fixation, reduction in the form of carbon chains, mainly in the form of cellulose-lignin-hemicellulose in the cell walls. Therefore, if the objective is energy transformation (“energy gain”), we should focus on the biomass, where energy is stored, which means mainly the transformation, i.e. release of the energy

László Márton, Miklós Gábor Fári

of the C-C bonds in the cell walls. In most of the current crops, the ligno-cellulose cell wall material is only a by-product (straw, stem, seed coat, processing waste), which is usually expensive due to the costs of collection and transport.

The contribution of biomass to the energy use in Hungary could be increased up to 80% from the present 10% if the suitable lands were used for biomass crop production.

The so-called third generation biomass crops were selected and bred for the purpose of intensive and environmentally-benign biomass crop production. As it cannot be avoided that biomass production should be limited to lands of marginal quality and should not compete with food production, drought tolerance or its opposite, tolerance of inundation and waterlogging can be an important aspect. Crops should be resistant to other extreme growing site conditions such as high salt content, extreme pH, chemical residues, heavy metal pollution, which do not allow for food crop production, or the crops grown there are not suitable for consumption. Only perennial crops can be taken into consideration, because the annual soil cultivation reduces CO₂ fixation, and the perennial herbaceous crops show considerable, continuous sustainability, can be of remediative/ameliorative nature (especially grasses) and can improve the lands of marginal quality.

How can GM techniques assist the sustainability of biomass industry? The significance of GM technology and its contribution to the breeding of optimum biomass crops and to their optimization for energy production can be enormous, because an extremely quick and effective breeding work is necessary due to the shortage of time. This justifies the use of new genetic-epigenetic modification/controlling GM and the somaclonal breeding techniques. The advanced state of recombinant DNA technologies enables an increasingly precise transformation of the genome and the fine tuning and adjustment of gene functions, with which the lack of century-long breeding work can be compensated.

Major R&D fields

For the use of GM technology, not only the easily and spectacularly changeable parameters should be considered such as the augmentation of fermentable sugar and starch content, the modification of starch structure and metabolism optimal for fermentation, where otherwise great advances have been made during the effort towards producing seed alcohol more efficiently.

Revolutionary successes have been achieved, when complex metabolic changes with a dramatic effect on cell wall composition and structure were created in GM biomass crops. On the one hand, a higher energy level was achieved by increasing e.g. the lignin content, which results in surplus energy on direct burning and pyrolysis. On the other hand, significant results were achieved in increasing the proportion of cellulose and hemi-cellulose, which are the substrates of fermentable sugar-release (chemical or enzymatic decomposition). These can

10. The possible role of GM technology in the breeding, propagation and processing of future biomass crops

be used for the production of ethanol by yeast fermentation, or of alcohols with longer carbon chains and various other products by other microbes. The significance of these longer carbon chained products is that they can be separated from water without the energy-consuming distillation, they can be easily purified and directly used as liquid fuel. At present the highest efficacy of ethanol extraction is achieved in steam-combustion systems producing 1 t alcohol from 4 tons of dry plant matter, i.e. 10 t alcohol per hectare for a local giant reed (Arundo donax L.) plantation (Figure 10.1.), which can reach up to 25–35 tons of ethanol under tropical and subtropical conditions. This is several times higher than the efficacy of seed alcohol or even sugarcane alcohol production. These data refer to the so-called “basic type” plants; it is not difficult to calculate how dramatic the impact of the introduction of new-generation GM plants on the ethanol market will be.

Figure 10.1. One of the most promising area of the modern plant

biotechnology is understanding the complex physiological and molecular regulation processes of somatic embryogenesis. Four-month- old giant reed (Arundo donax L.) syn-plant biomass plantation propagated from somatic embryos (Plants for the Future Experimental Garden, University of Debrecen, 2010)

Also very promising is the creation of GM plants, in which not only the accessibility and composition of the cell wall are changed, but the cellulose- decomposing enzyme is also produced by the plant, an enzyme which, under the appropriate conditions, can decompose the plant material to sugars. In other cases, the expensive decomposing enzymes were synthesized by plants and were added to the decomposing reaction. The excess “syngas” derived from pyrolysis of GM crops with higher lignin content and of lignin by-products, which is used directly for heating and electricity production, can be polymerized and converted into liquid fuel by modern variants of the old Fischer-Tropsch method.

Another field where the GM technology has made great contributions is the increase in crop size (since size and growth rate are of primary importance in biomass crop production). Giant sized plants were grown by modifying the regulation of the genes controlling hormone production and effect (e.g. ARGOS) and genes controlling cell division. The shift in the cell wall composition in GM crops resulted not only in more or less products (shift in the lignin/cellulose ratio), but also e.g. in better digestibility, which indicates a better availability of cellulose and hemicellulose for degradation to sugars.

The application of the so-called siRNA technology in GMOs started a revolution in the field of biomass energy. The significance of this technology in the regulation of metabolic processes and their dramatic transformation is comparable to that of the introduction of restriction endonucleases in recombinant DNA technology. In this way, multigenic functions can be regulated, blocked or adjusted as needed.

There has not yet been a breakthrough, but intensive research is carried out on the modification of photosynthesis mechanism in C3 and C4 plants, so that optimum water utilization could be reached in biomass energy crops. This is an extremely complex process, and there are still a lot of questions to be clarified at the level of basic research. The siRNA technology will definitely result in a revolution in this field, not the least because there are several multigenic functions in these processes.

As most of the biomass crops (GM or non-GM) are not suitable for further classical plant breeding (e.g. they are sterile, have no seeds or they are so aneuploid that they cannot be handled by sexual propagation), the only options are somaclonal breeding and clonal propagation. Prolonged clonal propagation can be frequently accompanied by clonal deterioration, which can be explained on the one hand by the accumulation of epigenetic changes and, on the other hand, by virus accumulation. However, in continuously maintained embryogenic cell cultures and plants regenerated therefrom, great homogeneity and stability can be observed, therefore if such cultures are used for producing GM plants and then mass micropropagation procedures are developed, the above disadvantages can be prevented. Clonal (epigenetic) ageing can be avoided and continuously rejuvenated plants can be planted.

Another alternative is classical vegetative propagation, which can be done only expensively or with difficulty in the case of large plants with rhizomes by chopping up of rhizomes or stem cuttings, and very frequently it does not yield viable propagation material (e.g. there is no viable bud on it, or it is virus-infected that can be transferred to other plants).

The road to producing future biomass crops will probably lead through programming the genes responsible for embryogenesis by using GM techniques, as it has been successfully done for certain model species. In such cases, the GM technique is an option but not always an essential method (Figure 10.2).

László Márton, Miklós Gábor Fári

10. The possible role of GM technology in the breeding, propagation and processing of future biomass crops

Figure 10.2. The Artificial Plant Ovary (APO) developed by the University

of Debrecen will generate promising technological break-throw in the large-scale clonal propagation practice of some GM-based industrial biomass crops (Populus sp, Eucalyptus sp., sugarcane, etc.)

A new propagation technology developed in recent years based on international co-operation and on an instrument, APO (Artificial Plant Ovary) developed by the Plant Biotechnology Department of the University of Debrecen enabled the mass propagation of Arundo donax, one of the most promising biomass crops, which produces record amounts of biomass under almost any climate condition. One of the special biological keys of the process was the controlled induction and maintenance of the continuous embryogenic state, whereas the other was the synchronized, homogeneous regeneration of such cultures into syn-plants. These new biotechnological tools resulted in a production system which meets all the requirements of large-scale production, the testing of which has started worldwide. This procedure can also be applied to other biomass crops; the elaboration of similar procedures is in progress e.g. for Malvaceae at the University of Debrecen.